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ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2012 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 820 Environmental Pesticide Exposure and Neurobehavioral Effects among Children of Nicaraguan Agricultural Workers TERESA RODRÍGUEZ ISSN 1651-6206 ISBN 978-91-554-8488-0 urn:nbn:se:uu:diva-182164
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ACTAUNIVERSITATIS
UPSALIENSISUPPSALA
2012
Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 820
Environmental PesticideExposure and NeurobehavioralEffects among Children ofNicaraguan AgriculturalWorkers
TERESA RODRÍGUEZ
ISSN 1651-6206ISBN 978-91-554-8488-0urn:nbn:se:uu:diva-182164
Dissertation presented at Uppsala University to be publicly examined in Frödingsalen,Ulleråkersvägen 40 A, Uppsala, Thursday, November 15, 2012 at 09:00 for the degree ofDoctor of Philosophy (Faculty of Medicine). The examination will be conducted in English.
AbstractRodríguez, T. 2012. Environmental Pesticide Exposure and Neurobehavioral Effects amongChildren of Nicaraguan Agricultural Workers. Acta Universitatis Upsaliensis. DigitalComprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 820. 66 pp.Uppsala. ISBN 978-91-554-8488-0.
Background: Children exposed to pesticides are susceptible for neurodevelopmentaldisruption. Data from developing countries are scarce.
Aim: Assessing long-term and recent pesticide exposure in Nicaraguan children in relationto parental pesticide use and examining potential associated neurobehavioral effects.
Methods: In the first study, pre- and post-spraying urinary residues of the chlorpyrifosmetabolite TCPY and diazinon metabolite IMPY were measured among 7 subsistence farmersand 10 plantation workers, and in one child per worker. In the second study, for 110 childrenin an agricultural village and 22 in a non-agricultural village, aged 7-9, parental pesticideuse was assessed by hours of spraying and kilograms of active ingredients during pre-and-postnatal time windows, as proxies for children’s long term pesticide exposures. Urinary TCPY,3-PBA (pyrethroid metabolite), and 2,4-D were determined in 211 samples of 74 children of theagricultural village. IQ components and total IQ (WISC-IV) were evaluated in all agriculturalvillage children. Behavior was evaluated with the Conners’ Teacher Rating Scale-Revised:Short. Multivariate linear regression models assessed associations between long-term and recentexposure to organophosphates and pyrethroids and cognitive and behavioral scales.
Results: In study 1, post-spraying urinary levels of pesticide metabolites of subsistencefarmers and their children were highly correlated (r=0.85), but not those of plantation workersand their children. In study 2, a wide range of exposures was reported by parents for all pesticidesand time windows. The median urinary TCPY (3.7 μg/g creatinine), 3-PBA (2.8), and 2,4-D(0.9) were comparable to other studies for TCPY and 3-PBA but high for 2,4-D. Maximumlevels were the highest reported for all compounds. Prenatal use of organophosphates affectedworking memory, and methamidophos also verbal comprehension and total IQ. Urinary TCPYwas associated with poorer working memory. Organophosphate exposures were not associatedwith children’s behavior. Pyrethroid exposure during the first year of life associated with poorerperceptual reasoning and behavior, and urinary 3-PBA with a number of cognitive functionsand ADHD in girls but not in boys.
Conclusion: Nicaraguan children in poor agricultural areas are highly exposed to pesticides,which is influenced by parental pesticide use in subsistence farms. Organophosphate andpyrethroid exposures adversely affect their neurobehavioral development.
Keywords: pesticides, organophosphates, pyrethroids, children, cognitive function, behavioraloutcomes, neurodevelopment
Teresa Rodríguez, Uppsala University, Department of Medical Sciences, Akademiskasjukhuset, SE-751 85 Uppsala, Sweden.
© Teresa Rodríguez 2012
ISSN 1651-6206ISBN 978-91-554-8488-0urn:nbn:se:uu:diva-182164 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-182164)
To my daughters María Teresa y Ana Lucía
My husband César Augustoand my parents Antonio y Teresa
With special dedication to My grandpa ”Papa Tomás”,
who is always alive in my heart
List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I Rodríguez, T., Younglove, L., Lu, C., Funez, A., Weppner, S.,
Barr, DB., Fenske, RA. (2006). Biological monitoring of pes-ticide exposures among applicators and their children in Nica-ragua. International Journal in Occupational and Environmen-tal Health 12, 312-320.
II Rodríguez, T., van Wendel de Joode, B., Lindh, CH., Rojas, M., Lundberg, I., Wesseling, C. (2012). Assessment of long-term and recent pesticide exposure among rural school chil-dren in Nicaragua. Occupational and Environmental Medicine 69, 119-125.
III Rodríguez, T., van Wendel de Joode, B., Hernández-Bonilla, D., García, L., Roque, E., Lundberg, I., Wesseling, C. Cogni-tive deficits in organophosphate exposed children of Nicara-guan subsistence farmers. Manuscript.
IV Rodríguez, T., van Wendel de Joode, B., Hernández-Bonilla, D., Soto, A., Balladares, S., Lundberg, I., Wesseling, C. Pyre-throids exposure and neurobehavioral performance in school age children in rural Nicaragua. Manuscript.
Reprints were made with permission from the respective publishers.
Contents
1. Introduction ............................................................................................... 11
2. Background ............................................................................................... 12 2.1 Pesticides in an agricultural setting in Nicaragua ............................... 12
2.1.1 High pesticide use ....................................................................... 12 2.1.2 Pesticide use in conditions of poverty ........................................ 13 2.1.3 Regulation of pesticide use in Nicaragua ................................... 13 2.1.4 Evidence of environmental contamination by pesticides ............ 14 2.1.5 Evidence of human pesticide exposure and health effects in agricultural areas .................................................................................. 14
2.2 Determinants and pathways of children’s pesticide exposure in agricultural families .................................................................................. 16 2.3 Neurotoxicity of pesticides ................................................................. 17
2.3.1 Mechanism of neurotoxicity for organophosphates and pyrethroids ........................................................................................... 17 2.3.2 Special vulnerability of children to toxic effects in the nervous system .................................................................................................. 18 2.3.3 Neurobehavioral effects of organophosphate and pyrethroid insecticides ........................................................................................... 19
3. Objectives ................................................................................................. 31
4. Methods .................................................................................................... 32 4.1 First data collection and paper I. ........................................................ 32
4.1.1 Study population ......................................................................... 32 4.1.2 Exposure assessment .................................................................. 32 4.1.3 Data analysis ............................................................................... 32
4.2 Second data collection and papers II, III and IV. ............................... 33 4.2.1 Study population ......................................................................... 33 4.2.2 Exposure assessment .................................................................. 33 4.2.3 Outcome assessment ................................................................... 35 4.2.4 Data analysis ............................................................................... 37
4.3 Ethical considerations ........................................................................ 38
5. Results ....................................................................................................... 39 5.1 Children’s pesticide exposure ............................................................ 39
5.1.1. Recent pesticide exposure (Papers I and II) ............................... 39 5.1.2 Children’s long term pesticide exposure (Paper II) .................... 40
5.2 Neurobehavioral effects in early school-age children in relation to organophosphate and pyrethroid exposure ............................................... 42
5.2.1 Children’s long term pesticide exposure and neurobehavioral effects (Paper III and Paper IV) ........................................................... 42 5.2.2 Children’s recent pesticide exposure and neurobehavioral effects (Paper III and Paper IV) ........................................................... 43
6. General discussion .................................................................................... 46 6.1 Main findings ..................................................................................... 46
6.1.1 Children’s pesticide exposure ..................................................... 46 6.1.2 Children’s pesticide exposure and neurobehavioral effects ........ 50
6.2 Validity ............................................................................................... 51
7. Conclusions ............................................................................................... 53
8. Implications .............................................................................................. 54
9. Acknowledgements ................................................................................... 55
10. References ............................................................................................... 58
Abbreviations
2,4-D: 2,4 dichlorophenoxyacetic acid AChE Acetylcholinesterase ADHD Attention deficit hyperactivity disorder ARYase Arylesterase ASQ Ages and stages questionnaires BARS Behavioral Assessment and Research System BCN Central Bank of Nicaragua (Banco Central de
Nicaragua) BHC Benzene hexachloride (gamma-hexachloro cy-
clohexane, Lindane) BMI Body Mass Index CBCL Child Behavior Checklist CCCEH Columbia Center for Children’s Environmental
Health. Children from a cohort of African Amer-ican and Dominican mothers in New York City
CENOP Center of neuropsychological testing evaluation and pedagogical orientation (Centre d’evaluation neuropsychologique et d’orientation pédago-gique)
CHAMACOS Center for the Health and Assessment of Mo-thers and Children of Salinas. Children from a cohort of Latina mothers in agricultural commu-nities in California
CTRS-R:S Revised Conners’ Teachers Rating Scale, short version
DAPs Dialkylphosphates DBCP 1,2-dibromo-3-chloropropaneDDT DichlorodiphenyltrichloroethaneDMAP Dimethyl alkyl phosphate
DQs Children developmental quotientsdw dry weightU.S. EPA United States Environmental Protection Agency FAO Food and Agriculture OrganizationGABA Gamma aminobutyric acid
GDS Gesell Developmental SchedulesICBF Icon-calendar-based formIMPY 2-isopropoxy-4-methyl-pyridinolINIDE National Information and Development Institute
(Instituto Nacional de Información de Desarrollo)
IQ Full-Scale intelligence quotient K-CPT Conners’ Kiddie Continuous Performance Test MDI Mental Development Index MINSA Health Minister (Ministerio de Salud) MSCEHC Mount Sinai Children’s Environmental Health
Center
NBAS Neonatal Behavioral Assessment Scale NCTB Neuropsychological core test battery
NEPSY-II Neuropsychological AssessmentOPs OrganophosphastesPDI Psychomotor Development Index
PON1 Paraoxonase 1
PSI Processing Speed IndexPENTB Pediatric Environmental Neurobehavioral Test
BatteryPON 1 Paraoxonase 1PRI Perceptual Reasoning Indexp,p’- DDE p,p’- dichlorodiphenyldichloroethylenep,p’- DDT p,p’- dichlorodiphenyldichloroethaneTCPY 3,5,6-trichloro-2-pyridinolUNAN National Autonomous University of Nicaragua
(Universidad Nacional Autónoma de Nicaragua) VCI Verbal Comprehension Index
WHO World Health OrganizationWISC-IV Wechsler Intelligence Scale for Children, 4th
edition WISC-R Wechsler Intelligence Scale for Children, revised WMI Working Memory Index
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1. Introduction
In Nicaragua, agriculture is the most important economic activity. Use of toxic pesticides is widespread and high, both on plantations and among subsistence farmers (Corriols, 2009). With 42% of the population living in rural areas (INIDE, 2012), pesticide exposures and subsequent health effects have been a serious concern since decades (Corriols, 2009). Rural children are an especially important target group for research on pesticide hazards and risks. There exists evidence, from studies in the United States, that children in agricultural communities have more contact with pesticides in comparison with children from urban areas, through a number of different pathways (Karr, 2012). In addition, recent studies, mostly from industrialized countries, are increasingly showing that pesticide exposure early in life can disrupt the normal development of the central nervous system causing impairments in children’s cognitive and psychomotor performance (London et al., 2012).
In developing countries in general, and in Nicaragua specifically, very few studies have addressed pesticide exposures among children and their determinants. Nonetheless, important exposures occur in Nicaraguan children (Dowling et al., 2005). No previous studies have addressed long-term health outcomes from pesticide exposures among Nicaraguan children.
This thesis intends to fill some of the knowledge gaps by examining pesticide exposures in children of Nicaraguan farmers and plantation workers, both recent exposures by means of urinary residue analyses (papers I and II) and cumulative exposures by means of indices of parental pesticide use as proxies of children’s long-term pesticide exposures during vulnerable prenatal and postnatal time-windows of development (paper II). This thesis then explores associations between these children’s exposures to common neurotoxic pesticides and their performance on cognitive and behavioral tests, specifically for organophosphates (paper III) and pyrethroids (paper IV).
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2. Background
2.1 Pesticides in an agricultural setting in Nicaragua 2.1.1 High pesticide use In Nicaragua, with six million inhabitants, over 40% of the economically active population works in agriculture (INIDE, 2012). From the very beginning of introduction of pesticides on the market, in the 1950s, the agriculture in Nicaragua has been highly dependent on pesticides (FAO, 2012). Pesticide use started in cotton cultivation with the introduction of the dichlorodiphenyltrichloroethane (DDT) and, shortly after, the extremely toxic organophosphate insecticide methyl parathion. The use of insecticides in cotton plantations was massive. For example, 454 tons of methyl parathion were applied in 1952 (15 kg/Ha) (Duarte, 2004). DDT and carbamate insecticides were authorized in that same period by the Ministry of Health for malaria vector control (MINSA, 2001).
During the next two decades, besides the high use in cotton cultivation, new export crops (coffee, banana, sugarcane and tobacco) and the expansion of subsistence crops (beans, corn, rice, soy bean and vegetables) forced dramatically the increase of pesticide imports. The use of organochlorine insecticides (DDT, aldrin, chlordane, toxaphene, DBCP, BHC, endrin, mirex) and organophosphates (methyl parathion, malathion) was especially intensive (Duarte, 2000).
The analysis of import data evidences the continued use of organophosphates and carbamates classified by the World Health Organization (WHO) as class 1A (extremely hazardous) and 1B (highly hazardous). In the context of international regulations, at the end of the 1990s the use of pesticides gradually shifted from persistent (organochlorines) and highly toxic pesticides (carbamates and organophosphates) to less persistent and less acutely toxic pyrethroids (MAGFOR 2001; MAGFOR, 2004).
Today, Nicaragua imports just over ten thousand tons of active ingredient of pesticides, equivalent to 0.8 kg active ingredient per inhabitant per year in rural Nicaragua. Twenty-two percent of the import volume corresponds to highly or extremely highly hazardous pesticides from an acute toxicity pers-pective and almost 30% are neurotoxic (Bravo et al., 2011).
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2.1.2 Pesticide use in conditions of poverty Inadequate handling during preparation and application of pesticides by agri-cultural workers in Nicaragua has been repeatedly reported (Corriols, 2009; Aragón et al, 2001; Blanco et al, 2005; Rojas et al. 2009). The following reported practices could directly influence children’s exposure to pesticides: storage of the chemicals under beds, in the kitchen or in places of easy access for children; inappropriate use of empty pesticide containers to store water, vegetable oils or food; deficient personal hygiene, washing of work clothes together with the clothes of the family, and dumping of empty pesticide containers near playing areas.
Besides inappropriate pesticide handling, rural families usually live in the middle of or close to the cultivated fields. The homes are very often one-room houses with dirt-floors, roofs in very poor condition and completely open to cultivated fields (Figure 1) (Rojas et al., 2009). In consequence, drifting of pesticides from the sprayed crops and contamination of indoor environments is highly probable.
Figure 1 One room-houses in Los Zanjones /Calle Real where this thesis was carried out. Areas where families expend most of the time are open to the cultivated fields.
2.1.3 Regulation of pesticide use in Nicaragua In 1998, Law 274 was approved, the basic law for the regulation and control of toxic and hazardous pesticides and other high risk substances. This law also determines that the Ministry of Agriculture and Forestry (MAGFOR) is the authority in charge of its implementation. MAGFOR has restricted and banned a number of hazardous pesticides over the last two decades. Currently, uses of terbufos, carbofuran, methomyl, methyl parathion, methami-dophos, etoprophos, endosulfan, chlorpyrifos, paraquat and aluminum phosphide are restricted, whereas all uses of aldicarb, chlordane, heptachlor, chlordimeform, DBCP, DDT, aldrin, dieldrin, endrin, EDB, HCH/BHC, lindane, pentachlorophenol, toxaphene, 2,4,5-T and monocrotophos are cancelled (MAGFOR 2001; MAGFOR, 2004). Despite
14
that Nicaragua has a fairly good pesticide legislation and the government assumedly follows international guidelines for pesticides regulation, such as the FAO Code of Conduct, structures and resources to ensure compliance are limited.
2.1.4 Evidence of environmental contamination by pesticides The combination of high use of pesticides, precarious handling of pesticides, and poor regulatory implementation gives rise to frequent events of environmental pollution, which have been documented in Nicaragua since de-cades. The studies of environmental pesticide contamination have focused on water, sediments and, more recently, soil in agricultural areas. No studies in air have been carried out yet.
A screening of persistent chlorinated hydrocarbons was carried out in 1995 in the main lagoons on the Pacific coast of Nicaragua, where cotton cultivations predominated. In sediments, very high concentrations of total DDTs (270 ng g-1 dw) and toxaphene (420 ng g-1 dw) were found. Other compounds such as arochlors, chlordane, endosulfan, and dieldrin were also identified (Carvahlo et al., 1999). In water, the pesticides determined were dichlorvos (up to 410 ng g-1 dw), diazinon (up to 150 ng g-1 dw), chlorpyrifos (up to 83 ng g-1 dw), toxaphene (up to 17,450 ng g-1 dw), total DDTs up to 478 and arochlor up to 119 (Carvalho et al., 2002). In soils, many organochlorines were also identified with concentrations of most of these compounds being below 5 ng g-1, but concentrations of toxaphene being extremely high (17000- 44000 ng g-1 dw) (Carvahlo et al., 2003).
A study carried out in 1999 in Posoltega, the same area where a part of this thesis was carried out, reported dieldrin, p,p’- DDE, fenthion, ethylparathion, methomyl and carbaryl in soils; and dieldrin, p,p’- DDE, DDT and cya-nazine in water in relation to cotton cultivation (Cuadra et al.,1999). In Río San Juan, the river that forms the border with Costa Rica, p,p’- DDE, lindane and p,p’- DDT were found in water, and dieldrin, p,p’- DDE and lindane in suspended particles, in relation to sesame, cane sugar and rice crops (PROCUENCA San Juan, 1997). Montenegro et al., (2007) showed hexachlorobenzene, dieldrin and DDT in well waters in villages close to banana plantations.
2.1.5 Evidence of human pesticide exposure and health effects in agricultural areas Exposures to pesticides and related health effects have been documented in Nicaragua essentially in adult agricultural workers. Determinants of dermal
15
exposure have been studied in subsistence farmers (Blanco et al., 2005) and measured with a visual semi-quantified fluorescent method (Aragón et al, 2006). Extensive dermal contamination post-application, especially on hands, arms, feet and back, was observed for most farmers. All the 32 agricultural workers participating in this study had at least one contaminated body part and no body part was free from contamination in all workers (Aragón et al., 2005).
Acute pesticide poisonings have been reported in relation to organophosphate and carbamate insecticides and the herbicide paraquat (Amador et al., 1993; McConnell et al., 1990; Rosenstock et al., 1991; McConnell and Hruska, 1993; Keifer et al., 1996 (a), Corriols et al., 2009).
In Nicaraguan agricultural workers with previous acute organophosphate poisoning, persistent neurological effects have been reported. Compared to a never poisoned control group, poisoned agricultural workers performed worse on five of six subtests of the World Health Organization Neuropsychological Core Test Battery (WHO-NCTB) and on additional tests that assessed together verbal and visual attention, visual memory, visuomotor speed, sequencing and problem solving, and motor steadiness and dexterity, two years after an episode of acute pesticide poisoning (Rosenstock et al., 1991). Of the workers previously poisoned with methamidophos, 25% had abnormally high vibrotactile thresholds (McConnell et al., 1994).
In a cohort study that followed men after poisoning with an organophosphate pesticide starting at hospital discharge, grip and pinch strength were impaired at two years of follow up among men severely poisoned with methamidophos and chlorpyrifos both occupationally and intentionally (Miranda et al., 2004). Decreased visuomotor performance and a marked increase in neuropsychiatric symptoms were also reported two years post-poisoning (Delgado et al., 2004).
In children, organophosphate exposure has been documented by means of decreased cholinesterase activity (Keifer et al., 1996 (b); McConnell et al., 1999) and urinary levels of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCPY) (Dowling et al., 2005). Between 1995 and 2006, the pesticide poisoning surveillance system registered 2069 acute pesticide poisonings in children aged 5 to 14. Of these, 47% were caused by organophosphate pesticides, predominantly methamidophos and chlorpyrifos (Corriols and Aragon, 2010). Long-term health effects have not been investigated in children in Nicaragua.
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2.2 Determinants and pathways of children’s pesticide exposure in agricultural families Determinants of exposure refer to factors that influence exposure in a broad sense, from proximal factors such as behavior, hygiene, weather or landscape up to more distal social, economic and climatic factors. The term ‘exposure pathway’ refers to the course of a contaminant from its origin to its endpoint, considering release at the source (for example a sprayed agricultural field), environmental fate and transport mechanisms through water, air, soil, and biota; the point of exposure or the place where someone can get in contact with the contaminant in the environment (for example home dust), the route of uptake (inhalation, ingestion or dermal contact), and the exposed receptor population (children in our case). Pathways of exposure characte-rize how children can come into contact with a pesticide. Knowledge on determinants and pathways, which interlink and overlap, is important in designing and implementing effective preventive and control measures.
In industrialized countries pathways for children’s pesticide exposures in agricultural areas have been studied. Pathways have, for example, identified carry-home of pesticides by farmer and farmworker parents on their conta-minated skin, clothes and shoes (Gomes et al., 1999; Simcox et al.,1995; Bradman et al., 1997; Loewenherz et al.,1999; Fenske et al., 2000; Lu et al., 2000; Mc Couley et al., 2001; Curl et al., 2002; Thompson et al., 2003; Coronado et al., 2006) and contamination of domestic dust (Simcox et al., 1995, Lu et al., 2000, Mc Couley et al., 2001). Of special importance is transfer of pesticides to the unborn child in pregnant women. Between 25 to 60% of pregnant farmworkers of the CHAMACOS cohort in Salinas Valley California incurred in risky behaviors with regard to basic norms of hygiene such as hand washing, bathing, and use of protective clothing, storage and washing of clothes, house cleaning, bringing home fruit and vegetables directly from the field, and wearing work clothes more than 30 minutes after work (Goldman et al., 2004).
In Posoltega, the same rural area where the fieldwork for papers II, III and IV was carried out, a parallel qualitative assessment of pesticide exposure of the children evidenced factors that indirectly influenced pesticide use by the parents and others that directly determined exposure of the children (Rojas et al., 2009). This study found that the economic dependence on farming for family subsistence and absence of feasible alternatives of employment, the presence of big peanut plantation around the villages with high volumes of pesticide use, the reluctance of poor farmers to use alternative pest control methods out of fear for crop loss, and child labor as a traditional part of the upbringing, were important determinants of children’s exposures to
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pesticides. Poverty is the underlying determinant of these interconnected co-mmunity and family factors for contamination and exposures. In addition, the weak governmental enforcement of pesticide legislation, especially concerning working conditions and environmental contamination in relation to economically powerful export plantations (in this case peanut plantations surrounding the study community), is a more distal determinant of exposure among these rural children. This study also identified pathways, i.e. how the pesticides came into contact with the children, including take home, environmental exposures and direct manipulation by the child.
2.3 Neurotoxicity of pesticides Organophosphate and pyrethroid insecticides directly target nervous tissue as their mechanism of toxicity.
2.3.1 Mechanism of neurotoxicity for organophosphates and pyrethroids Organophosphates The primary target of organophosphate insecticides (OP) is the enzyme acetylcholinesterase (AChE), which hydrolyzes the neurotransmitter acetylcholine (ACh) in both the peripheral and the central nervous system. The reaction between an OP and the active site in the AChE results in the formation of a temporary intermediate complex that is partially hydrolyzed leaving a phosphorylated (inactive) enzyme. Under normal conditions, AChE can be reactivated only at a very slow rate (Bjørling-Poulsen et al., 2008). Some OPs have the ability to bind persistently to the active site of AChE to produce an irreversibly inhibited enzyme by a mechanism known as aging (Pope et al., 1999). The excessive amount of undestroyed ACh cause nicotinic and muscarinic signs and symptoms. Nicotinic manifestations, occu-rring in severe cases and late in the course, comprise fasciculations, cramps, weakness, hypertension, tachycardia, mydriasis, pallor and neuromuscular paralysis. Muscarinic manifestations include bradycardia, hypotension, rhinorrhea, bronchorrhea, bronchospasm, cough, severe respiratory distress, hypersalivation, nausea and vomiting, abdominal pain, diarrhea, fecal incontinence, urinary incontinence, blurred vision, miosis, increased lacrimation and diaphoresis (Singh and Sharma, 2000).
In addition to excitatory effects in both central and peripheral nervous system, ACh also plays a key role in regulation of morphogenetic cell movements, cell proliferation, growth, and differentiation in the development brain. The interference in the cholinergic system by OPs may
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have morphological, neurochemical and functional effects (Lauder and Schambra 1999). Cell signaling cascades that control neural cell replication and differentiation appear to be among the most sensitive targets for OPs developmental neurotoxicity and, in particular, the regulation of cyclic AMP (cAMP). Animal studies confirm that inhibition of cell signaling cascades in developing brain, disrupts the processes of replication and differentiation of neurons, axonogenesis and synaptogenesis (Schuh et al., 2002; Yanai et al.,2002; Meyer et al., 2003, 2004, 2005; Curtin et al., 2006).
Pyrethroids The effects of pyrethroids on the central and peripheral nervous system are complex and information regarding the potential developmental neurotoxicity of this class of compounds is limited. Effects in the nervous system in development have been associated with altered structure and function of voltage-gated sodium channels (Marban et al., 1998). The pyrethroids affect both the activation (opening) and inactivation (closing) of the channel, resulting in a hyperexcitable state producing abnormal repetitive nerve impul-ses (Ginsburg and Narahashi, 1993; Narahashi et al., 1998). Pyrethroids type I (without alfa-cyano-3-phenoxybenzyl moiety) hold sodium channels open for a relatively short time period (milliseconds) and type II (with alfa-cyano-3-phenoxybenzyl moiety) keep the channel open for a prolonged time period (up to seconds) (Soderlund, 2012). Additional mechanism for developmental neurotoxicity may involve antagonism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in chloride channels, (Abalis et al., 1986; Soderlund, 2012), modulation of nicotinic cholinergic transmission, enhancement of noradrenalin release, and direct actions on voltage dependent calcium or chloride ion channels (Soderlund et al., 2002). It is difficult to know the implications of these mechanisms in the process of maturation of nervous system because insufficient information is available in the scientific literature; the animal studies were conducted with inadequate study design, used formulated products, and/or had inadequate control group (Shafer et al., 2005).
2.3.2 Special vulnerability of children to toxic effects in the nervous system The special vulnerability of children to effects of pesticides is due to both physiological and behavioral reasons. On the one hand, there are important differences in physiological characteristics between children and adults. Young children have a larger skin surface area relative to body weight, hig-her basal metabolic rate, and greater oxygen requirements; they also eat more food, drink more water per body weight and breathe more air than adults; children’s ability to metabolize, detoxify and excrete toxic
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compounds is also immature (Selevan et al., 2000). On the other hand, some children’s behavior, such as the hand to mouth behavior, playing on the ground or playing with empty pesticide containers or in the crop fields make them more susceptible to enter in contact with pesticides (Freeman et al., 2001; Freeman et al., 2005; Black et al., 2005). Finally, because children have more years of life ahead than most adults do, they have more time to develop chronic effects that may be initiated by early exposures (Landrighan et al., 1999).
The nervous system is in continuous process of maturation concluding well into adulthood, on average at the age of 27 (Needleman, 2006). Some maturation periods are most susceptible to the toxic damage of environmental exposures. An important window for development of cognitive and behavioral function is the maturation of the frontal lobe, which is not complete until the age of 6, which is actually the reason that the inclusion of the children to the formal education is set at this age (Needleman, 2006). Research to evaluate neurological effect of environmental exposures in early stages in the life should consider this time point (Lanphear et al., 2005; Needleman, 2006; Weiss, 2000; Weiss et al., 2004; Weiss and Bellinger, 2006)
2.3.3 Neurobehavioral effects of organophosphate and pyrethroid insecticides A series of studies have shown a relationship between exposure to pesticides during pregnancy or early years of life and neurodevelopmental effects. Table 1 summarizes current evidence regarding effects in neurodevelopment in children exposed to organophosphates and pyrethroid insecticides. The evidence mainly came from three cohort studies in the United States: A cohort of children of Latina mothers’ in agricultural communities in Salinas Valley California (CHAMACOS); a cohort of children of African American and Dominican mothers in New York City (CCCEH) and a multiracial cohort of children born in The Mount Sinai Hospital studied by the Mount Sinai Children’s Environmental Health Center (MSCEHC). .
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Tab
le 1
Chi
ldre
n’s
pest
icid
e ex
posu
re a
nd n
euro
beha
vior
al e
ffect
s: a
rev
iew
of s
tudi
es fr
om J
anua
ry 2
001
to J
une
2012
.
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
USA
(R
auh
et a
l.,
2012
)
Lon
gitu
dina
l bir
th
coho
rt /
40
(C
CC
EH
)
5.9
- 1
1.2
year
s
Chl
orpy
rifo
s /
chlo
rpyr
ifos
in
umbi
lica
l cor
d bl
ood
Mag
netic
re
sona
nce
imag
ing
Hig
h ch
lorp
yrif
os
expo
sure
was
ass
ocia
ted
with
enl
arge
men
t of
cort
ical
reg
ions
rel
ated
w
ith
atte
ntio
n, r
ecep
tive
la
ngua
ge, s
ocia
l co
gniti
on, e
mot
ions
and
in
hibi
tory
con
trol
. C
hina
(G
uodo
ng e
t al
., 2
012)
Cro
ss-s
ectio
nal /
301
23
- 2
5 m
onth
s
Org
anop
hosp
hate
s /
DA
P c
once
ntra
tions
in
chi
ldre
n’s
urin
e sa
mpl
es
DQ
s ba
sed
on
GD
S
No
asso
ciat
ion
was
fo
und
betw
een
child
ur
inar
y le
vels
of
DA
Ps
and
any
of th
e D
Qs
scor
es.
USA
(B
ouch
ard,
et
al.,
2011
)
Lon
gitu
dina
l bir
th
coho
rt /
329
(C
HA
MA
CO
S)
7 ye
ars
O
rgan
opho
spha
tes
/ D
AP
con
cent
ratio
ns
in u
rine
col
lect
ed
duri
ng p
regn
ancy
an
d fr
om c
hild
ren
at 6
mon
ths
and
1,
2, 3
.5 a
nd 5
yea
rs
of a
ge
WIS
C-I
V
A
vera
ged
mat
erna
l DA
P
conc
entr
atio
ns w
ere
asso
ciat
ed w
ith
poor
er
scor
es f
or W
MI,
PS
I,
VC
I, P
RI,
and
Ful
l-S
cale
IQ
.
21
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
USA
(R
auh
et a
l.,
2011
)
Lon
gitu
dina
l bir
th
coho
rt /
265
(C
CC
EH
)
7 ye
ars
C
hlor
pyri
fos
/ ch
lorp
yrif
os in
um
bili
cal c
ord
bloo
d
WIS
C-I
V
F
or e
ach
stan
dard
de
viat
ion
incr
ease
in
expo
sure
(4.
61 p
g/g)
, F
ull-
Sca
le I
Q d
ecli
ned
by 1
.4%
, and
WM
I de
clin
ed b
y 2.
8%.
U
SA
(Eng
el e
t al.,
20
11)
Lon
gitu
dina
l bir
th
coho
rt /
20
0: 1
2 m
onth
s 27
6: 2
4 m
onth
s 16
9: 6
to 9
yea
rs
( M
SC
EH
C)
12 m
onth
s 24
mon
ths
6 -
9 ye
ars
Org
anop
hosp
hate
s /
DA
Ps
in m
ater
nal
urin
e sa
mpl
es;
PO
N1a
ctiv
ity a
nd
PO
N1
poly
mor
phis
m
BS
ID-I
I fo
r ch
ildre
n of
12
and
24 m
onth
s an
d W
PPS
I-II
I fo
r ch
ildre
n 6
to 9
ye
ars
Dec
rem
ent i
n m
enta
l de
velo
pmen
t at 1
2 m
onth
s am
ong
blac
ks
and
His
pani
cs.
Ass
ocia
tions
app
eare
d to
be
enh
ance
d am
ong
child
ren
of m
othe
rs w
ho
carr
ied
the
PO
N1
Q19
2R
gen
otyp
e.
In s
choo
l age
chi
ldre
n,
pren
atal
tota
l dia
lkyl
- an
d di
met
hylp
hosp
hate
m
etab
olite
s w
ere
asso
ciat
ed w
ith
decr
emen
ts in
per
cept
ual
reas
onin
g in
the
mat
erna
l P
ON
1 Q
192R
ge
noty
pe.
USA
L
ongi
tudi
nal b
irth
36
mon
ths
Per
met
hrin
/ B
SID
-II
N
o as
soci
atio
ns w
ith
22
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
(Hor
ton
et a
l.,
2011
) co
hort
/
342
for
per
met
hrin
in
pers
onal
air
27
2 fo
r pe
rmet
hrin
in
plas
ma
230
subj
ects
for
pi-
pero
nyl b
utox
ide
in
pers
onal
ai
r (C
CC
EH
)
perm
ethr
in le
vels
m
easu
red
in
mat
erna
l and
um
bili
cal c
ord
plas
ma
coll
ecte
d on
de
liver
y;
perm
ethr
in a
nd
pipe
rony
l but
oxid
e le
vels
mea
sure
d in
pe
rson
al a
ir
coll
ecte
d du
ring
pr
egna
ncy
perm
ethr
in
Pip
eron
yl b
utox
ide
in
pers
onal
air
sam
ples
(>
4.34
ng/
m3 )
scor
ed 3
.9
poin
ts lo
wer
on
the
MD
I th
an th
ose
wit
h lo
wer
ex
posu
res.
Phi
lippi
nes
(Ost
rea
Jr. e
t al
., 20
11)
Lon
gitu
dina
l bir
th
coho
rt /
754
2 ye
ars
P
yret
hroi
ds,
carb
amat
es a
nd
orga
noph
osph
ates
/ pr
opox
ur, c
yflu
-th
rin,
chl
orpy
rifo
s,
cype
rmet
hrin
, pr
etila
chlo
r,
bioa
lleth
rin,
m
alat
hion
, dia
zino
n an
d tr
ansf
luth
rin
in
mat
erna
l hai
r an
d bl
ood,
infa
nt’s
hai
r,
cord
blo
od a
nd
mec
o-ni
um
Gri
ffit
hs m
enta
l de
velo
pmen
tal
scal
e
Exp
osur
e to
pro
poxu
r w
as n
egat
ivel
y re
late
d to
m
otor
dev
elop
men
t, bu
t w
as u
nrel
ated
to s
ocia
l an
d pe
rfor
man
ce d
eve-
lopm
ent.
N
o si
gnif
ican
t re
latio
nshi
ps b
etw
een
pyre
thro
ids
and
neur
odev
e-lo
pmen
t at 2
4 m
onth
s w
ere
foun
d.
23
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
USA
(E
sken
azi e
t al.,
20
10)
Lon
gitu
dina
l bir
th
coho
rt /
353
(C
HA
MA
CO
S)
2 ye
ars
O
rgan
opho
spha
tes
/ D
AP
con
cent
ratio
ns
in m
ater
nal u
rine
du
ring
pre
gnan
cy;
PO
N1 1
92 a
nd
PO
N1 –
108 g
enot
ypes
in
mot
hers
and
ch
ildr
en, a
nd a
ryl-
este
rase
(A
RY
ase)
an
d pa
raox
onas
e
(PO
ase)
in p
regn
ant
mot
hers
, cor
d an
d 2
year
s ol
d bl
ood.
MD
I an
d PD
I of
th
e B
ayle
y S
cale
s an
d C
BC
L
Chi
ldre
n w
ith th
e P
ON
1 –10
8T a
llel
e ha
d po
orer
M
DI
scor
es a
nd
som
ewha
t poo
rer
PDI
scor
es.
Ass
ocia
tion
bet
wee
n D
AP
s an
d M
DI
scor
es
was
str
onge
st in
chi
ldre
n w
ith
PO
N1 –
108T
all
ele.
USA
(M
arks
et a
l.,
2010
)
Lon
gitu
dina
l bir
th
coho
rt/
654
331:
3.5
yea
rs
323:
5 y
ears
(CH
AM
AC
OS)
3.5
year
s
5 ye
ars
Org
anop
hosp
hate
s /
DA
P c
once
ntra
tions
in
mat
erna
l uri
ne
duri
ng p
regn
ancy
CB
CL
Vis
ual a
tten
tion
su
btes
t of
NE
PSY
-II
K-C
PT
Hill
side
Beh
avio
r R
atin
g S
cale
Pre
nata
l DA
Ps
wer
e no
t as
soci
ated
wit
h m
ater
nal
repo
rt o
f at
tent
ion
prob
lem
s an
d A
DH
D a
t ag
e 3.
5.
Pre
nata
l DA
Ps
wer
e as
soci
ated
wit
h m
ater
nal
repo
rt o
f at
tent
ion
prob
lem
s an
d A
DH
D a
t ag
e 5.
Pre
nata
l DA
Ps
wer
e
24
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
as
soci
ated
wit
h sc
ores
on
the
K-C
PT
(8%
of
chil
dren
sco
red ≥
70%
on
the
stan
dard
ized
A
DH
D C
onfi
denc
e In
dex
scal
e).
Som
e ef
fect
s ar
e st
rong
er
in b
oys
than
in g
irls
.
USA
(B
ouch
ard
et
al.,
2010
)
Cro
ss s
ectio
nal
/ 113
9
8 -
15 y
ears
Org
anop
hosp
hate
s /
DA
P c
once
ntra
tions
in
uri
ne o
f ch
ildre
n
Dat
a of
the
Nat
iona
l Hea
lth a
nd
Nut
ritio
n E
xam
inat
ion
Sur
vey
(200
0 –
2004
)
A s
truc
ture
d in
terv
iew
w
ith a
par
ent t
o as
cert
ain
AD
HD
di
agno
stic
sta
tus
A 1
0-fo
ld in
crea
se in
D
MA
P co
ncen
trat
ion
was
ass
ocia
ted
wit
h an
od
ds r
atio
of
1.55
for
A
DH
D.
Ecu
ador
(H
arar
i et a
l.,
2010
)
Cro
ss-s
ectio
nal /
87
Chi
ldre
n at
ten-
ding
Ecu
ador
ian
seco
nd a
nd th
ird
grad
e
Org
anop
hosp
hate
s /
mat
erna
l int
ervi
ew,
DA
Ps
in u
rine
and
er
ythr
ocyt
e ac
etyl
chol
ines
tera
se
activ
ity in
chi
ldre
n
Fin
ger
Tap
ping
T
ask,
San
ta A
na
For
m B
oard
, K-
CP
T (
vers
ion
5),
Cop
ying
Tes
t of
the
Sta
nfor
d-B
inet
(4
th e
diti
on),
R
aven
’s C
olor
ed
Chi
ldre
n w
ith p
rena
tal
expo
sure
fro
m m
ater
nal
gree
nhou
se w
ork
show
ed
cons
iste
nt d
efic
its
for
mot
or s
peed
(Fi
nger
T
appi
ng T
ask)
, mot
or
coor
dina
tion
(San
ta A
na
For
m B
oard
),
25
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
Pro
gres
sive
M
atri
ces,
and
D
igit
Spa
n of
W
ISC
-R
visu
ospa
tial p
erfo
rman
ce
(Sta
nfor
d-B
inet
Cop
ying
T
est)
, and
vis
ual m
emor
y (S
tanf
ord-
Bin
et C
opyi
ng
Rec
all T
est)
. C
urre
nt e
xpos
ure
(DA
PS
an
d ac
etyl
chol
ines
tera
se)
did
not a
ffec
t the
ou
tcom
es.
Ecu
ador
(H
anda
l et a
l, 20
08)
Cro
ss-s
ectio
nal /
121
3
- 23
mon
ths
A
ny p
esti
cide
/ oc
cupa
tiona
l hi
stor
y of
mot
hers
Pre
hens
ion
(rea
ch-a
nd-g
rasp
) an
d vi
sual
ski
lls
Chi
ldre
n w
hose
mot
hers
w
orke
d in
the
flow
er
indu
stry
(ex
pose
d to
pe
stic
ides
) du
ring
pr
egna
ncy,
sco
red
low
er
on c
omm
unic
atio
n an
d fi
ne m
otor
ski
lls a
nd h
ad
a hi
gher
odd
s of
hav
ing
poor
vis
ual a
cuit
y.
USA
(S
anch
ez-
Liz
ardi
et a
l.,
2008
)
Cro
ss-s
ectio
nal /
48
7 ye
ars
Org
anop
hosp
hate
s /
DA
Ps
in c
hild
ren’
s ur
ine
Sho
rt f
orm
of
the
WIS
C-T
hird
E
ditio
n,
Chi
ldre
n’s
Mem
ory
Sca
le;
Wis
cons
in C
ard
Sor
ting;
Tra
il M
akin
g T
est A
&
Hig
her
OP
pes
ticid
e m
etab
olite
con
cent
ratio
n le
vels
wer
e si
gnif
ican
tly
corr
elat
ed w
ith p
oore
r pe
rfor
man
ce o
n so
me
subt
ests
of
the
Wis
cons
in
Car
d S
orti
ng T
est
26
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
B; C
BC
L/4
-18
and
the
Tea
-che
r R
epor
t For
m
Bra
zil
(Eck
erm
an e
t al
., 20
07)
Cro
ss-s
ectio
nal /
56
10
- 1
8 ye
ars
Any
pes
tici
de
/chr
onic
pes
ticid
e ex
posu
re in
dex
BA
RS
Impa
irm
ent o
f ta
ppin
g,
digi
t spa
n, a
nd s
elec
tive
atte
ntio
n
USA
(E
ngel
et a
l.,
2007
)
Lon
gitu
dina
l bir
th
coho
rt /
311
(
MS
CE
HC
)
Neo
nate
s
O
rgan
opho
spha
tes
/ D
AP
s in
mat
erna
l ur
ine;
PO
N1
activ
ity
Bra
zelt
on N
BA
S
DA
Ps
rela
ted
to in
crea
se
in th
e nu
mbe
r of
ab
norm
al r
efle
xes,
and
in
crea
se in
the
prop
orti
on
of c
hild
ren
with
mor
e th
an th
ree
abno
rmal
re
flex
es.
The
re w
as a
str
ong
inte
ract
ion
betw
een
PO
N1
expr
essi
on le
vels
an
d to
tal d
imet
hyl a
lkyl
ph
osph
ates
on
risk
of
abno
rmal
ref
lexe
s.
USA
(E
sken
azi e
t al.,
20
07)
Lon
gitu
dina
l bir
th
coho
rt /
6
mon
ths
(n =
396
) 12
mon
ths
(n =
395
) 24
mon
ths
(n =
372
)
6, 1
2 an
d 24
m
onth
s O
rgan
opho
spha
tes
/ si
x no
nspe
cifi
c D
AP
met
abol
ites
, m
alat
hion
/ sp
ecif
ic
met
abol
ite
MD
A,
CB
CL
A
t 24
mon
ths:
Bay
ley
MD
I R
isk
of P
erva
sive
D
evel
opm
enta
l Dis
orde
r
27
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
(CH
AM
AC
OS)
an
d ch
lorp
yrif
os /
spec
ific
met
abol
ite
TC
PY
in p
regn
ant
mot
hers
and
chi
ld
urin
e E
cuad
or
(Han
dal e
t al.,
20
07)
Cro
ss-s
ectio
nal /
283
3
- 61
mon
ths
A
ny p
estic
ide
/ C
omm
unity
of
resi
denc
e
ASQ
D
efic
its o
n gr
oss
and
fine
mot
or a
nd s
ocio
-in
divi
dual
ski
lls
USA
(R
ohlm
an e
t al.,
20
07)
Cro
ss-s
ectio
nal /
175
11
9 ad
oles
cent
s an
d ad
ults
wor
king
in
agri
cult
ure
56
not
wor
king
in
agri
cult
ure
Adu
lts 2
8 ±
7.6
year
s
Ado
lesc
ents
15.
7 ±
1.6
year
s
Any
pes
tici
de /
year
s w
orki
ng in
ag
ricu
ltur
e.
Fin
ger
tapp
ing,
di
git s
ymbo
l, si
mpl
e re
acti
on
tim
e, d
igit
span
, pr
ogre
ssiv
e ra
tio,
sele
ctiv
e at
tent
ion,
ser
ial
digi
t lea
rnin
g,
cont
inuo
us
perf
orm
ance
, m
ath-
to-s
ampl
e,
reve
rsal
lear
ning
.
In a
dult
s an
d ad
oles
cent
s lo
w le
vels
of
pest
icid
es
over
man
y ye
ars
of
agri
cult
ural
wor
k ar
e as
so-c
iate
d w
ith
neur
olog
ical
impa
irm
ent
as m
easu
red
by th
e se
lect
ive
atte
ntio
n, d
igit
sym
bol,
and
reac
tion
tim
e te
sts.
Yea
rs o
f ex
peri
ence
han
dlin
g pe
stic
ides
wer
e in
vers
ely
asso
ciat
ed w
ith
defi
cits
in
neu
robe
havi
oral
pe
rfor
man
ce.
U
SA
(Rau
h et
al.,
L
ongi
tudi
nal b
irth
co
hort
/ 2
54
12, 2
4 an
d 36
m
onth
s C
hlor
pyri
fos
/ ch
lorp
yrif
os le
vels
in
PD
I an
d M
DI
of
Bay
ley
Sca
les
H
ighl
y ex
pose
d ch
ildr
en
scor
ed
28
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
2006
) (C
CC
EH
) um
bili
cal c
ord
plas
ma
CB
CL
on a
vera
ge 6
.5 p
oint
s lo
wer
on
the
PD
I an
d 3.
3 po
ints
low
er o
n th
e M
DI
and
had
mor
e at
tent
ion
prob
lem
s, A
DH
D, a
nd
perv
asiv
e de
velo
pmen
tal
diso
rder
pro
blem
s at
3
year
s of
age
com
pare
d w
ith
thos
e w
ith
low
er
leve
ls o
f ex
posu
reE
cuad
or
(Gra
ndje
an e
t al
., 2
006)
Cro
ss-s
ectio
nal /
79
7 ye
ars
Org
anop
hosp
hate
s /
eryt
hroc
yte
AC
hE
activ
ity a
nd u
rina
ry
excr
etio
n of
DA
Ps
in u
rine
of
child
ren.
San
ta A
na F
orm
B
oard
, WIS
C-R
, S
tanf
ord-
Bin
et
copy
ing
subt
est,
Fin
ger
tapp
ing
task
, R
eact
ion
time
test
Mot
her’
s oc
cupa
tion
in
flor
icul
ture
ass
ocia
ted
with
def
icits
on
visu
o-sp
atia
l and
res
pons
e sp
eed.
D
AP
met
abol
ites
as
soci
ated
wit
h re
acti
on
tim
e de
fici
ts.
Isra
el
(Kof
man
et a
l.,
2006
)
Cro
ss-s
ectio
nal /
52
26
exp
osed
26
une
xpos
ed
6 -
12 y
ears
O
rgan
opho
spha
tes
/ o
rgan
opho
spha
te
pois
oned
dur
ing
infa
ncy
vs
kero
sene
poi
sone
d vs
mat
ched
un
pois
oned
con
trol
s
Neu
rops
ycho
logi
cal
bat
tery
D
efic
its o
n lo
ng-t
erm
m
emor
y,
lear
ning
, inh
ibito
ry
mot
or c
ontr
ol
29
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
USA
(Y
oung
et a
l.,
2005
)
Lon
gitu
dina
l bir
th
coho
rt /
381
(CH
AM
AC
OS)
≤ 2
mon
ths
Org
anop
hosp
hate
s /
urin
ary
leve
ls o
f D
AP
s in
mat
erna
l ur
ine
at 1
4 an
d 26
w
eeks
of
preg
nanc
y an
d 7
days
pos
t-pa
rtum
.
Bra
zelt
on N
BA
S
Pre
nata
l exp
osur
e w
as
rela
ted
with
an
incr
ease
in
num
ber
of a
bnor
mal
re
flex
es a
nd m
ore
than
th
ree
abno
rmal
ref
lexe
s.
USA
(R
ohlm
an e
t al,
2005
)
Cro
ss-s
ectio
nal /
78
48
- 7
1 m
onth
s
Any
pes
ticid
e /
Agr
icul
tura
l co
mm
uniti
es v
s no
n-ag
ricu
ltura
l co
mm
uniti
es
Fin
ger
tapp
ing
Mat
ch-t
o-sa
mpl
e C
hild
ren
from
ag
ricu
ltura
l com
mun
ities
pe
rfor
med
poo
rer
on
mea
sure
s of
res
pons
e sp
eed
(Fin
ger
tapp
ing)
an
d la
tenc
y (M
atch
-to-
sam
ple)
com
pare
d to
ch
ildre
n of
non
-ag
ricu
ltura
l co
mm
uniti
es.
U
SA
(Ruc
kart
et a
l.,
2004
)
Cro
ss-s
ectio
nal /
226
10
7 ex
pose
d
119
unex
pose
d
6 ye
ars
M
ethy
l par
athi
on /
urin
ary
para
-ni
trop
heno
l lev
els
in c
hild
ren
and
envi
ronm
enta
l wip
e sa
mpl
es f
or m
ethy
l pa
rath
ion.
PE
NT
B
H
igh
expo
sure
to M
ethy
l pa
rath
ion
was
ass
ocia
ted
with
poo
r pe
rfor
man
ce in
ta
sks
invo
lvin
g sh
ort-
term
mem
ory
and
atte
ntio
n.
30
Cou
ntr
y (r
e-fe
ren
ce)
Stu
dy d
esig
n /
stud
y p
opu
lati
on
Ch
ild
ren
’s a
ge a
t n
euro
beh
avio
ral
test
ing
Pes
tici
de
/ ex
pos
ure
as
sess
men
t
Too
ls f
or
outc
ome
asse
ssm
ent
Neu
rob
ehav
iora
l eff
ects
Hig
h ex
posu
re w
as
defi
ned
as h
ouse
hold
M
ethy
l par
athi
on
≥ 1,
000 μg
/100
cm
2
or a
uri
nary
PN
P
leve
l ≥ 3
00 p
pbU
SA
(Roh
lman
et a
l.,
2001
)
Cro
ss-s
ectio
nal /
147
96
ado
lesc
ents
cu
rren
tly
wor
king
in
agri
cult
ure
51
ado
lesc
ents
cu
rren
tly
non-
mig
rato
ry
and
not w
orki
ng in
ag
ricu
ltur
e
13 -
18
year
s
Any
pes
ticid
e /
occu
patio
nal
expo
sure
BA
RS
D
efic
its o
n re
spon
se
spee
d,
Atte
ntio
n an
d co
mpl
ex
func
tioni
ng
AD
HD
: A
tten
tion
def
icit
hyp
erac
tivi
ty d
isor
der;
AC
hE:
acet
yl c
holi
nest
eras
e; A
RY
ase:
Ary
lest
eras
e; A
SQ
: A
ges
and
stag
es q
uest
ionn
aire
s; B
AR
S:
Beh
a-vi
oral
Ass
essm
ent
and
Res
earc
h S
yste
m;
BS
ID-I
I: B
ayle
y S
cale
s of
Inf
ant
Dev
elop
men
t, ve
rsio
n II
; C
BC
L:
Chi
ld B
ehav
ior
Che
ckli
st;
CC
CE
H:
Col
umbi
a C
ente
r fo
r C
hild
ren’
s E
nvir
onm
enta
l H
ealt
h. C
hild
ren
from
a c
ohor
t of
Afr
ican
Am
eric
an a
nd D
omin
ican
mot
hers
in
New
Yor
k C
ity;
CH
AM
AC
OS:
Cen
ter
for
the
Hea
lth
and
Ass
essm
ent o
f M
othe
rs a
nd C
hild
ren
of S
alin
as. C
hild
ren
from
a c
ohor
t of
Lat
ina
mot
hers
in a
gric
ultu
ral c
omm
unit
ies
in C
alif
orni
a; D
AP
s:
Dia
lkyl
phos
phat
es;
DM
AP
: D
imet
hyl
alky
l ph
osph
ate;
DQ
s: C
hild
ren
deve
lopm
enta
l qu
otie
nts;
GD
S:
Ges
ell
Dev
elop
men
tal
Sch
edul
es;
IQ:
Ful
l-S
cale
in
tell
igen
ce q
uoti
ent;
K-C
PT
: C
onne
rs’
Kid
die
Con
tinu
ous
Per
form
ance
Tes
t; M
DI:
Men
tal
Dev
elop
men
t In
dex;
MS
CE
HC
: M
ount
Sin
ai C
hild
ren’
s E
nvir
onm
enta
l H
ealt
h C
ente
r; N
BA
S:
Neo
nata
l B
ehav
iora
l A
sses
smen
t S
cale
; N
EP
SY
-II:
Neu
rops
ycho
logi
cal
Ass
essm
ent;
PD
I: P
sych
omot
or D
evel
opm
ent
Inde
x; P
EN
TB
: P
edia
tric
Env
iron
men
tal
Neu
robe
havi
oral
Tes
t B
atte
ry;
PO
N1:
Par
aoxo
nase
1;
WIS
C-I
V:
Wec
hsle
r In
tell
igen
ce S
cale
for
Chi
ldre
n, 4
th
edit
ion;
WIS
C-R
: Wec
hsle
r In
tell
igen
ce S
cale
for
Chi
ldre
n, r
evis
ed; W
PP
SI-
III:
Wec
hsle
r P
resc
hool
and
Pri
mar
y S
cale
of
Inte
llig
ence
- T
hird
Edi
tion
.
31
3. Objectives
The overall objective of this thesis was to examine the exposure to pesticides in Nicaraguan children in relation to parental pesticide use, and the occurrence of associated neurobehavioral effects, in a developing country context.
The specific objectives were
• To measure current pesticide exposure of children in rural Nicaragua in relation to parental pesticide use.
• To construct retrospective pre and postnatal quantitative indices of parental pesticide use in rural Nicaragua as proxies for children’s life-long pesticide exposures.
• To explore the relationships between pre and postnatal exposure to organophosphate and pyrethroid insecticides and cognitive performance and behavior in school-aged rural Nicaraguan children.
• To explore the relationships between recent exposure to organophosphate and pyrethroid insecticides and cognitive performance and behavior in these children.
32
4. Methods
This thesis is based on two separate data collections. The first study was conducted in 2003 and assessed exposures to the organophosphate insecticides chlorpyrifos and diazinon among pesticide applicators and their children. The second study, carried out during 2007-2008, assessed children’s long-term pesticide exposures through indices of parental pesticide use and children’s recent exposure through urinary residue levels, and examined associations between long-term and recent pesticide exposures with neurobehavioral effects.
4.1 First data collection and paper I. 4.1.1 Study population The study population included farm workers and their children from two areas in Nicaragua: 7 smallholders using the organophosphate chlorpyrifos on corn crops in León, and 10 workers on two banana plantations in Chinandega who sprayed diazinon. One child of each agricultural worker, between 2 and 12 years of age and residing with the applicator, was included in the study.
4.1.2 Exposure assessment Urine samples were collected from each pesticide applicator and from his child. A complete set of urine samples included four voids on the day of the application (before and immediately after spraying, at midday, and at the end of the workday), and three voids the day after the application (morning, midday and afternoon) for the sprayers and their children.
4.1.3 Data analysis Concentrations of 3,5,6-trichloro-2-pyridinol (TCPY), the specific metabolite of chlorpyrifos, and 2-isopropoxy-4-methyl-pyridinol (IMPY), the specific metabolite of diazinon, were log-normally distributed and the geometric mean, range and percentiles were calculated. Linear regression was used to examine the relationship between adult and child metabolite levels.
33
4.2 Second data collection and papers II, III and IV. 4.2.1 Study population This cross-sectional study was conducted in the northwest of Nicaragua. The study initially targeted the populations of children aged 7 to 9, attending grade 1 to 3, of two communities, an agricultural community versus a non-agricultural community. The communities were Los Zanjones/Calle Real in Posoltega, a highly agricultural region that produces peanut for exportation and cereals and vegetables for subsistence, and Colonia 20 de Julio in Vi-llanueva, also a rural area with predominantly informal nonagricultural commercial activity. In the agricultural community, 110 children were in-cluded in the study population (96% response) and in the non-agricultural community 74 (88% response). All children from Los Zanjones/Calle Real had a parent working in agriculture at the time of the study. In Colonia 20 de Julio no agricultural activity was registered since one year before the study, but 22 parents had been employed earlier in large scale agriculture.
The three papers used different subsets of these 284 children. The con-struction of life-long exposure indices, reported in Paper II, was based on data for all children of fathers with a pesticide use history from both com-munities (n=132). During the fieldwork, however, important differences between the two communities became apparent, which could influence the neurobehavioral performance of children. To avoid confounding from socio-economic disparities and differences in quality of schooling, the neurobeha-vioral assessment was restricted to the children of the agricultural communi-ty (n=110) who had similar socioeconomic backgrounds and all went to the same school (Papers III and IV). Recent exposure and associated neurobe-havioral effects were examined in a subset of the children of the agricultural community (n=74) (Papers II, III and IV).
4.2.2 Exposure assessment Long-term pesticide exposure Due to Hurricane Felix, no pesticides were applied in the agricultural com-munity at the time of neurobehavioral testing in 2007, and, therefore, no recent exposure occurred. An icon-calendar-based form (ICBF) (Engel et al., 2001; Monge et al., 2004; Monge et al., 2007) was used to interview the parents about children’s prenatal and life-time pesticide exposures. The ICBF was applied to parents of all 110 children of the agricultural communi-ty and to 22 parents of the children in the non-agricultural community with a history of agricultural work in the past. Some children were siblings and only one ICBF was applied to their parent, but their individual data differed for exposure windows. The ICBF covered pesticide use data from one year before birth throughout pregnancy up to the current year. Stickers were used
34
to indicate life events and work history, to facilitate recall of pesticide use. The data collected were from one year before birth to the year of interview (2007). In the ICBF we registered data on pesticide use for different crops and years, specific trade names or active ingredients, dose, number of appli-cations during the spraying season, and usual hours of spraying. All farmers used manual backpack sprayers and some of the fathers had worked on plan-tations with tractor spraying. None had ever used protective equipment.
At the time of the interviews, most (n=74 ) were subsistence farmers spraying with backpacks, 28 were plantation workers spraying with tractor, and 8 were plantation workers who also owned a piece of land. Indices for pesticide use were constructed for the fathers and one widow mother. In some subsistence farming families (n=9) mothers also had participated in pesticide related tasks. For subsistence farming, a pesticide specific use in-dex of total kg used in a lifetime was constructed: dose (g/ha) * # ha * fre-quency (# application days). For plantation work we could not collect data on pesticide dose, and therefore the pesticide index was calculated as total number of hours in a lifetime: # application days * # hours per day. The la-tter was also used as a second index for subsistence farmers to be able to combine the groups. When fathers were both plantation worker and subsist-ence farmer simultaneously, their indices of hours of spraying were summed. Indices were constructed for five specific known neurotoxic pesticides (chlorpyrifos, methamidophos, cypermethrin, deltamethrin and 2,4-D), chemical groups of organophosphates, carbamates and pyrethroids, and for total pesticide use, by adding specific pesticide use scores.
Recent exposure In 2008, pesticide residue analyses in urine samples was added to the expo-sure assessment strategy to assess recent pesticide exposure of the children in relation to parental applications. However, also in 2008, only about half of the farmers were cultivating crops, this time due to delays in delivery of the seeds by the state agencies.
Effective urine sampling was difficult, because the fathers often changed their pesticide spraying plans at the last moment. Therefore, mothers filled in information in a calendar about the use of pesticide during the week of full moon, when farmers usually spray. At the same time, the children were asked to bring in a urine sample daily from Monday to Saturday. Samples were then related to parental pesticide applications with the help of the moon calendar. A complete set of urine samples for children, whose father had sprayed pesticides, consisted in four voids, one per day during 4 days (the day before application, the day of application, and 24 and 48 hours after application). Of the children, whose fathers did not spray pesticides, one or two voids were randomly selected during this sampling week. A total of 211 urine samples were collected for 74 children, 154 samples from 32 children
35
of fathers cultivating agriculture produce and 57 samples of 42 children of fathers without agriculture activity.
The urine samples were analyzed for the chlorpyrifos metabolite TCPY, the general pyrethroid metabolite 3-phenoxybenzoic acid (3-PBA), and 2,4-D according to Lindh et al. (2008) adjusted for urinary creatinine levels. Creatinine levels were analyzed using an enzymatic method described by Mazzachi et al., (2000).
4.2.3 Outcome assessment Neurobehavioral tests for children were selected based on the recommenda-tions of a workshop in Costa Rica, in February 2006, with participation of experts from North and Latin America (UNA, 2006). The testing was done at the schools of the communities, by psychologists trained and supervised by a neuropsychologist.
Cognitive performance of the children was evaluated with the Wechsler Intelligence Scale for Children (WISC IV) (Weschler, 2003). Twelve sub-tests grouped in four cognitive indices and total IQ were applied (Table 2).
Table 2 Cognitive indices and sub-test of WISC IV (Anderson et al., 2001; Braga and Campoz Da Paz; Wechsler, 2007)
Cognitive indices Functions assessed Sub-tests Verbal Compre-hension Index (VCI)
o ability to listen to a question,
o draw upon learned information from both formal and informal education,
o reason through an answer, and ex-press their thoughts aloud.
Similarities Vocabulary Comprehension
Perceptual Rea-soning Index (PRI)
o ability to examine a problem
o draw upon visual-motor and visual-spatial skills
o organize their thoughts, create solu-tions, and then test them.
Block design Picture concepts Matrix reasoning Picture comple-tion
Working Memory Index (WMI)
o ability to memorize new information, hold it in short-term memory
o manipulate the information to pro-duce some result or reasoning pro-cesses.
Digit Span Letter-number sequencing
Processing Speed Index (PSI)
o ability to focus attention and quickly scan
o discriminate between, and sequential-ly order visual information
o persistence and planning ability
Coding Symbol search Cancellation
36
Behavior of the children was measured using the revised Conner’s Teachers Rating Scale short version (CTRS-R:S). The CTRS-R:S is a commonly used measure of behavioral problems in children. Several abbreviated forms exist; in this thesis the revised short-form of 28 items was used (Conners, 1997). Questions were responded on a 4-point scale: ‘‘never’’ (0), “occasionally” (1), “often” (2) and ‘‘very often’’ (4) and the answers were grouped in three scales (Oppositional, Cognitive Problems/Inattention, and Hyperactivity). In addition, the test yields an ‘ADHD Index’, which partially overlaps with the hyperactivity scale. Raw scores were calculated by summation of points for each question comprised in every sub-scale.
In addition we applied a test for general intelligence of the mother, inde-pendent of language and reading and writing skills (Raven, 2003) and a child development questionnaire (CENOP FL, 2012). A complete physical and neurological examination was conducted, and children’s height and weight and body mass index (BMI) were registered.
An overview of the periods of data collection, study populations, and ex-posure and outcome assessment in studies 1 (Paper I) and 2 (Paper II, III and IV) is presented in Table 3.
Table 3. Overview of the study methods
2003 Paper I
2007 Paper II, III and IV
2008 Paper II, III and
IV Study population
-10 banana plantation worker and child pairs -7 farmer and child pairs
Initial study population: 110 children of an agricultural community and 74 children of a non-agricultural com-munity (including 22 with history of parental pesticide use in agriculture) -Paper II: 132 children with a parental history of pesticide use in agriculture (110 + 22) Paper III and IV: 110 children of the agricultural community
74 children of the ag-ricultural community
Exposure assessment
Urinary metabolites of fathers and children 221 urine samples -Urinary TCPY (chlorpyrifos metabolite) -Urinary IMPY (dia-zinon me-tabolite)
Long term exposure indices based on data collected by ICBF -Exposure indices in hours and kg of a.i for total pyrethroids and cyperme-thrin, and for total orga-nophosphates, chlorpyrifos and methamidophos. -Time windows: prenatal, first year of life, age over 1 to age in 2007.
Urinary metabolites -154 samples of 32 children whose par-ents were cultivating crops -57 samples of 42 children whose par-ents were not cultivat-ing crops -Urinary 3-PBA (general pyrethroid metabolite) -Urinary TCPY (chlorpyrifos metabo-lite)
37
2003 Paper I
2007 Paper II, III and IV
2008 Paper II, III and
IV Cognitive assessment
WISC-IV -Verbal Comprehension Index (VCI)
o Similarities o Vocabulary o Comprehension o Block design
-Perceptual Reasoning Index (PRI) o Picture concepts o Matrix reasoning o Picture completion
-Working Memory Index (WMI) o Coding o Symbol search o Cancellation
-Processing Speed Index (PSI) o Digit Span o Letter-number sequencing
-Total IQ
WISC-IV (shortened test bat-tery) Verbal comprehen-sion domain o -Similarities o -Vocabulary o -Comprehension
Perceptual reasoning domain o Matrix reasoning o Picture comple-
tion
Working memory domain o Coding o Symbol search
Processing speed domain o Digit span o Letter-number
sequencing
Beha-vioral assessment
CTRS-R:S -Oppositional -Cognitive problems/inattention -Hyperactivity -ADHD index
CTRS-R:S -Oppositional -Cognitive pro-blems/inattention -Hyperactivity -ADHD index
4.2.4 Data analysis Indices of pesticides use were normalized by log10 conversion. Because no normative data for cognitive and behavioral tests exist for Central American populations, the raw scores were used for sub-tests of WISC-IV. Linear re-gressions were performed to examine, for each time window, associations between indices of kg of a.i. and the outcome variables, adjusting for poten-tial confounders. In the models with urinary metabolites as the explanatory variable, the geometric mean and the maximum urinary TCPY for each child (1 to 6 samples) were used.
To construct the models, we evaluated whether potential confounders changed the crude regression coefficient by more than 10% in bivariate analyses. For the cognitive outcomes these covariates were children’s school year, maternal years of education, the Raven test score and age of children.
38
Maternal years of education and Raven test score were correlated (r= 0.59, p< 0.05) and also children’s school year and children’s age (r= 0.64, p< 0.01). We retained children’s school year and maternal years of education in the models for the cognitive outcomes, and excluded Raven test score and children’s age since they did not further influence the effect estimates. In the models for behavioral outcomes, covariates were children’s sex and age. Models for cognitive and behavioral outcomes in relation to organophos-phate exposures were also adjusted for life time indices of pyrethroid use, and viceversa. Models assessing effects from recent exposures were in addi-tion adjusted for lifetime exposure indices. Weight, height or body mass index as proxies for nutritional status, years of education of the father, fa-thers’ age, and mothers’ age had no effect on any of the coefficients and were therefore not included.
4.3 Ethical considerations Study 1 (Paper I) was approved by the Ethical Committee of the Faculty of Medical Sciences of UNAN-León (08-2003) and by the Human Subject Committee at the University of Washington (03-5936-E-01).
Study 2 (Paper II, III and IV) was approved by the Ethical Committee of the Faculty of Medical Sciences of UNAN-León (150-2007). At the start of the study, researchers informed participants, including children, about the objectives and characteristics of the study in writing as well as verbally, and all parents signed an informed consent form. Analysis of pesticide bi-omarkers in Sweden was approved by the regional ethical board at the Medi-cal Faculty, Lund University (Regionala Etikprövningsnämnden in Lund, dnr 208/2009).
39
5. Results
5.1 Children’s pesticide exposure 5.1.1. Recent pesticide exposure (Papers I and II) This thesis found evidence that children of agricultural families in Nicaragua are highly exposed to pesticides.
Pesticide residues in fathers and their children were found before and af-ter parental pesticide application (Paper I). Detectable levels of TCPY were found in 48 of 53 samples (91%) of small-scale farmers, and in 37 of 54 samples (69%) taken from their children. Concentrations of TCPY peaked in the fathers 27 hours post application (geometric mean 25.8 μg/L, a 30-fold increase, p<0.01) and at 8.5 hours in the children (geometric mean of 2.99 μg/L, a 3-fold increase p>0.05). A high correlation was observed between urinary levels of TCPY of fathers and their children in the evening samples of the day that chlorpyrifos was sprayed (beta = 1.29; CI = 0.19–2.90; R2 = 0.73; Pearson r= 0.85, p= 0.03). Although the geometric mean of urinary TCPY was clearly higher in adults than in children (25.8 μg/L vs 3.0 μg/L), the highest TCPY concentration observed among the children (125 μg/L) approached the highest concentration among the fathers (147 μg/L). The relationships between pesticide application by fathers and children’s expo-sure was less evident in plantation workers spraying diazinon, for whom the metabolite IMPY was found only in 3 of the 57 samples of their children. Instead, unexpectedly, TCPY was detected in 57 of 58 of the plantation workers’ samples (98%), and in 45 of 56 of their children’s samples (79%), ranging from non-detectable to 109 μg/L for adults and from non-detectable to 20.2 μg/L for their children.
Also in the agricultural community of subsistence farming surrounded by peanut plantations, measurable quantities of pesticide residues were found in the urine of all children with 96% of the children’s urine samples containing at least two and 71% containing all three residues that were analyzed (Paper II). The P50 for TCPY was 3.7 μg/g creatinine, for 3-PBA 2.8, and for 2,4-D 0.9; maximum levels were the highest reported in the literature for all three pesticides. TCPY peaked 1 day after application from 3.9 to 16.5 μg/g based on eight datasets and 3-PBA peaked 4 days after application from 2.6 to 3.8 μg/g based on 22 datasets. However, the highest residue levels, those over the 90th percentile, were observed predominantly in children whose parents
40
had not applied the pesticide in question: 18 of the 21 highest TCPY and 15 of the 21 highest 3-PBA.
5.1.2 Children’s long term pesticide exposure (Paper II) Parents of the 132 schoolchildren of the agricultural community and the nonagricultural community with a pesticide use history reported together 47 different active ingredients on the ICBF, including 34 organophosphate, carbamate and pyrethroid insecticides. For all pesticides, there was an ample gradient both in hours and dose of active ingredient used by the parent (Ta-ble 4).
Table 4. Parental use indices of five neurotoxic pesticides during their children’s life-time
Specific pesticide Index in hours Index in kg of a.i
Chlorpyrifos, median (min; max) 114 (2; 1584) 19.2 (0.37; 548)
Methamidophos, median (min; max) 84 (6; 1964) 12.2 (0.30; 780)
Cypermethrin, median (min; max) 81 (8; 1976) 2.1 (0.01; 354)
Deltamethrin, median (min; max) 40 (2; 265) 0.97 (0.04; 22.7)
2,4-D, median (min; max) 4.2 (2; 38,8) 8.8 (1.1; 22.7)
For these five pesticides, the median of kg of a.i sprayed per year by fathers were higher during the prenatal period and first year of life than other time windows. A pattern of change in the number of hours of application between periods was not so clear (Table 5).
41
Tab
le 5
. Med
ian
of h
ours
and
kg
of a
.i of
rel
evan
t neu
roto
xic
pest
icid
es u
sed
by ti
me-
win
dow
s
C
hlo
rpyr
ifos
M
eth
amid
opho
s C
yper
met
hri
n
Del
tam
eth
rin
2,
4-D
Ind
ices
in h
ours
Per
icon
cept
ion
and
preg
nanc
y (1
ye
ar)
39.0
43
.0
19.0
18
.0
16.0
Fir
st y
ear
of li
fe (
1 ye
ar)
19.0
22
.0
22.0
23
.0
24.0
Age
1 to
5 (
aver
age
per
year
, 4
year
s)
16.0
16
.0
6.0
4.0
12.0
Fro
m a
ge 6
(av
erag
e pe
r ye
ar, u
p to
200
7, 2
to 4
yea
rs)
40.0
20
.0
8.0
8.0
6.0
Ind
ices
in k
g of
a.i
Per
icon
cept
ion
and
preg
nanc
y 7.
0 4.
0 0.
7 1.
4 5.
5
Fir
st y
ear
of li
fe (
1 ye
ar)
3.8
2.4
0.6
0.8
3.0
Age
1 to
5 (
aver
age
per
year
, 4
year
s)
1.3
1.0
0.2
0.2
0.4
Fro
m a
ge 6
(av
erag
e pe
r ye
ar, u
p to
200
7,
2.0
1.4
0.3
0.4
0.9
42
5.2 Neurobehavioral effects in early school-age children in relation to organophosphate and pyrethroid exposure 5.2.1 Children’s long term pesticide exposure and neurobehavioral effects (Paper III and Paper IV) Cognitive effects In general, a poorer performance of children highly exposed to organophos-phate and pyrethroids on most of sub-test of WISC-IV was observed. The prenatal index in kg of total use of organophosphates was associated with lower scores in Verbal Comprehension Index (VCI), Perceptual Reasoning Index (PRI), Working Memory Index (WMI), and total IQ. The adjusted beta indicated that the Working Memory Index score decreased 3.2 points for each 10-fold increase in kg of total organophosphate used by fathers, and similar decreases were observed in working memory index with the specific OPs chlorpyrifos and methamidophos. The prenatal index for methamidophos was also associated with decreased scores on the compre-hension and perceptual reasoning indices and on total IQ.
The index in hours for pyrethroid use during the first year of life was sig-nificantly associated with poorer performance on the Perceptual Reasoning Index (PRI), with the PRI score decreasing 1.1 point for each 10-fold in-crease in hours of pyrethroid use. No associations were observed between hours of pyrethroid spraying by the fathers and the indices for verbal com-prehension, working memory and processing speed, or for total IQ for any of the time windows.
No significant associations were found between exposure indices of or-ganophosphates and pyrethroids for the age older than 1 and cognitive per-formance. No relevant differences between sexes were observed in cognitive assessment in relation to exposure to pesticides.
Effects in behavior The organophosphate exposure indices in kg of a.i in none of the time win-dows associated with the scales of the CTRS-R:S. Regarding pyrethroids, we observed increasing scores on the hyperactivity subscale and ADHD index scores with increasing hours of use, during the first year of life as well as in the time windows after age one.
The association between parental pesticides use indices as a proxy of children long-term exposure and neurobehavioral outcomes is summarized in Figure 2.
43
5.2.2 Children’s recent pesticide exposure and neurobehavioral effects (Paper III and Paper IV) Cognitive effects After adjusting for confounders a 10-fold increase in urinary residue values of the chlorpyrifos metabolite TCPY was related with a non-significant 3.2 points decrease on the Working Memory Index, significantly for a subtest of this domain, Letter-number sequencing.
A poorer performance on most cognitive sub-test was noted with increas-es in 3-PBA urinary residue levels. Scores on Vocabulary (verbal compre-hension), Picture Completion (perceptual reasoning) and Letter-number Se-quencing (working memory) decreased 2.9, 2.4 and 0.4 points, respectively, in association with 10-fold increases in urinary levels of 3-PBA. Different effect patterns occurred for boys and girls. For boys, scores on the Vocabu-lary and Coding tests decreased significantly with increasing 3-PBA, where-as for girls scores on the Symbol search (processing speed).
Effects in behavior TCPY urinary values were not related to scales of the Conners’ Teacher Rating Scale. With regard to 3-PBA no behavioral effects were seen in boys but ADHD scores in girls increased 8.5 points for each 10 folds increase in 3-PBA urinary levels.
The association between urinary pesticides metabolites and neurobehav-ioral outcomes is summarized in Figure 3.
44
F
igur
e 2.
Ass
ocia
tion
s be
twee
n lo
ng-t
erm
pes
tici
de e
xpos
ure
indi
ces
and
cogn
itiv
e an
d be
havi
oral
per
form
ance
am
ong
Nic
arag
uan
scho
ol
child
ren.
45
F
igur
e 3.
Ass
ocia
tion
s be
twee
n ur
inar
y pe
stic
ide
met
abol
ites
and
cog
niti
ve a
nd b
ehav
iora
l per
form
ance
am
ong
Nic
arag
uan
scho
ol c
hild
ren.
46
6. General discussion
6.1 Main findings This thesis examined the exposure to pesticide in Nicaraguan children in relation to parental pesticide use, and the occurrence of associated neurobe-havioral effects, in a developing country context. In summary we reported evidence that children of agricultural families are highly exposed to pesti-cides and this exposure is influenced by occupational activities of fathers as well as by environmental pesticide contamination. The main cognitive ef-fects from long-term pesticide exposure were a poorer performance in work-ing memory in association with prenatal kg of organophosphate sprayed by fathers, specifically chlorpyrifos and methamidophos, poor performance in verbal comprehension and total IQ scores associated with prenatal kg of methamidophos, and poor performance in perceptual reasoning with hours of use of pyrethroids during the first year of life. Urinary TCPY was also, not significantly, related to working memory but it was not associated with be-havioral outcomes. Urinary 3-PBA was associated negatively with a number of cognitive functions and with ADHD in girls but not in boys.
6.1.1 Children’s pesticide exposure The observed urinary levels of pesticide metabolites in children evidence that children of agricultural families in Nicaragua are highly exposed to pes-ticides. Although the geometric mean of TCPY were similar to the findings in other studies, the geometric mean of 3-PBA and the maximum for both TCPY and 3-PBA are the highest reported in the international literature for children (Figure 4). Comparison with pesticide levels in other studies that have evaluated cognitive and/or behavioral outcomes was difficult, because these studies used other pesticide biomarkers, such as urinary dialkyl-phosphates (Guodong et al., 2012; Bouchard et al., 2011; Engel et al., 2007), chlorpyrifos in umbilical cord blood (Rauh et al., 2012, 2011, 2006) or spe-cific pesticides measured in maternal blood or meconium (Ostrea Jr et al., 2011; Horton et al., 2011).
The long-term exposure indices cannot be contrasted with other studies because the only previous study that has utilized data of parents’ pesticide use collected with ICBF as a proxy of children’s exposures, did not publish
47
quantitative summaries of pesticide use and only reported the association between these data and childhood leukemia (Monge, 2006).
It seemed that pesticides exposure in children from agricultural areas is influenced by fathers’ occupation. A take-home pathway was evidenced by the correlation observed between TCPY levels in adult and child samples taken in the evening of the chlorpyrifos’ application day reported (Paper I) and the increasing urinary levels of pesticide metabolites on the days follow-ing parental application (Paper II). These results are consistent with other reports of take-home (Gomes et al., 1999, Simcox et al., 1995, Bradman et al., 1997, Loewenherz et al.,1999, Fenske et al., 2000, Lu et al., 2000, Mc Couley et al., 2001). However, it is evident that other pathways besides the take-home occurred in our study population. For example, the highest levels of urinary pesticides metabolites were mostly unrelated to any pesticide ap-plication by the parents suggesting the possibility of pesticides drifting from neighboring plantations or small cultivated fields of neighbors, as well as domestic pesticide use.
The parallel qualitative study in the same agricultural area where the data collection for this thesis was conducted, mentioned previously in section 2, identified through interviews and focus group discussions important path-ways of children’s exposure (Rojas et al., 2009). The pathway of environ-mental exposures appeared in association with pesticide applications in the proximity of houses and schools, in particular due to the drift of pesticide used in peanut plantations. Take-home pathway was also important in rela-tion to the parents’ behavior after pesticide applications such as getting home with their contaminated clothes, tools and application equipment, the use of leftovers of agricultural pesticides for domestic pest control, the storage of pesticides inside their homes, and the inadequate disposal and/or re-use of empty pesticide containers. Direct manipulation of pesticides by the child also occurred, which was gender-differentiated. Boys helped their fathers in the fields with pesticide application. Girls helped their mothers in cleaning activities at home, doing laundry of contaminated work clothes, and with domestic pest control. The qualitative information about pathways and routes of pesticide uptake is summarized in in Table 6.
48
F
igur
e 4.
Com
pari
son
with
oth
er s
tudi
es o
f th
e ge
omet
ric
mea
ns o
f th
e ur
inar
y ch
lorp
yrif
os’
met
abol
ite
3,5,
6-tr
ichl
oro-
2-py
ridi
nol (
TC
PY
) an
d th
e no
n-sp
ecif
ic p
yret
hroi
d m
etab
olite
3-p
heno
xybe
nzoi
c ac
id
(3-P
BA
.)
49
Tab
le 6
. Pat
hway
s of
chi
ldre
n’s
pest
icid
e ex
posu
re a
mon
g ru
ral N
icar
agua
n ch
ildre
n
Pat
hw
ay
Sou
rce
of c
onta
min
atio
n E
nvi
ron
men
tal
sub
stra
te f
or
cont
amin
ant
tran
spor
tati
on
Poi
nt
of
exp
osu
re
Rou
te o
f ex
po-
sure
R
ecep
tor
pop
ula
tion
En
viro
nm
enta
l P
estic
ide
appl
icat
ion
in s
urro
undi
ng p
ea-
nut p
lant
atio
n P
esti
cide
use
in s
mal
l far
ms
Air
W
ater
D
ust
Sur
face
s
In th
e ho
useh
olds
A
t sch
ool
In th
e fi
elds
Inha
latio
n D
erm
al c
onta
ct
Inge
stio
n
Boy
s an
d gi
rls
Tak
e-h
ome
D
omes
tic
appl
icat
ion
of le
ftov
ers
of a
gri-
cultu
ral p
estic
ides
E
mpt
y pe
stic
ide
cont
aine
rs
Pes
ticid
es s
tore
d in
side
hom
e P
aren
ts’
cont
amin
ated
clo
thes
Air
D
omes
tic
dust
In th
e ho
useh
olds
Inha
latio
n D
erm
al c
onta
ct
Boy
s an
d gi
rls
Gir
ls
Ch
ild la
bor
Pes
ticid
e ap
plic
atio
n eq
uipm
ent
Rec
entl
y sp
raye
d fi
eld
Dom
estic
pes
ticid
e us
e C
onta
min
ated
dus
t and
sur
face
s at
hom
e P
aren
ts’
cont
amin
ated
clo
thes
and
sho
es
Air
S
urfa
ces
Air
W
ater
S
urfa
ces
In th
e fi
elds
In
the
hous
ehol
ds
In th
e fi
elds
Inha
latio
n D
erm
al c
onta
ct
Inha
latio
n D
erm
al c
onta
ct
Boy
s G
irls
50
6.1.2 Children’s pesticide exposure and neurobehavioral effects Effects from long-term exposures during vulnerable time windows The association between organophosphate exposure and cognitive effects found in this thesis is congruent with previous studies. Decrease in working memory performance and also full scale IQ, have been reported in associa-tion with maternal urinary dialkylphosphate levels (Bouchard et al., 2011; Engel et al., 2011) and chlorpyrifos levels measured in umbilical cord blood (Rauh et al., 2011). The poor performance in perceptual reasoning observed in relation to methamidophos has also been reported in relation to maternal urinary dialkylphosphate levels (Bouchard et al 2011; Engel et al., 2011). Some additional studies have reported cognitive impairment in association with organophosphate exposure but direct comparisons are difficult because other cognitive tools were used and/or other age groups were studied (Sanchez-Lizardi et al, 2008, Eskenazi et al., 2007; Handal et al., 2008; Ha-rari et al., 2010). However, regardless the age groups or the method for cog-nitive evaluation used, all findings are related with poor capacity of holding in mind and integrating information in memory.
The data presented here demonstrate an association between hours of use of pyrethroids during the first year of life and a poorer performance on per-ceptual reasoning. No other epidemiological studies have reported neurobe-havioral toxicity in relation to pyrethroids. One study examined whether permethrin levels, measured during pregnancy in mothers’ plasma and as individual airborne levels, as well as in cord blood affected children’s cogni-tion. No associations were reported with permethrin but decrease in cogni-tive performance was associated with piperonyl butoxide a synergist of pyre-throids insecticides (Horton et al., 2011), which could be also a proxy for pyrethroid exposures. Despite the almost total lack of epidemiologic evi-dence, animal studies have demonstrated neurotoxic effects of pyrethroids on the developing brain. In rats and mice exposed to pyrethroids during pregnancy and early postnatal days, hyperactivity and dose dependent de-creases in learning capacity have been reported (Shafer et al., 2005; Lazarini et al., 2001; Sinha et al., 2006).
Behavioral effects Unlike other studies, no behavioral effects in relation to pre or postnatal organophosphate exposures were detected in this study. Attention problems, attention-deficit/hyperactivity disorder problems, and pervasive develop-mental disorder problems have been reported in association with chlorpyri-fos concentrations in umbilical cord blood (Rauh et al., 2006), children’s urinary dialkyl phosphates (Bouchard et al., 2010) and maternal urinary di-alkyl phosphates (Eskenazi et al., 2007; Marks et al., 2010). A possible ex-planation is that Conners’ Behavior Rating Scale is self-reported by teachers and it is possible that some abnormal behaviors were not reported because
51
they are tolerable within the Nicaraguan cultural context. Nonetheless, be-havioral effects were observed for postnatal pyrethroid exposures. ADHD in girls but not in boys was associated with 3-PBA, in our knowledge no previ-ous studies have examined this potential association.
6.2 Validity In this thesis, misclassification of long-term pesticide exposure is a main concern for bias. Parents were asked to recall pesticide applications as far as 10 years back and, in consequence, it is likely that both omissions and over-estimations of dose and hours of spraying occurred. Farmers reported 47 different active ingredients on the IBCF during the lifetime of their children, but there were no governmental statistics on pesticide use available to check the validity of the data obtained from the questionnaires. The use of the ICBF for quantifying children’s life time exposure was extremely labor-intensive both with regard to applying the questionnaire and with systemati-zing the data for analyses. However, the ICBF has advantages that justify its use, since the farmers liked the method and did not get tired during the often rather prolonged interview. Overall, the recall of farmers seemed acceptable, particularly in the case of the “most used” pesticides.
The data on child exposure were derived from parental pesticide use data, which is only a proxy for their true exposures. However, the results of the assessment of children’s urinary residue pre and post parental pesticide ap-plication (paper I) evidenced a reasonable relationship between fathers’ pes-ticide use and children’s exposure among farmers, also in accordance with another Nicaraguan study (Dowling et al., 2005).
In addition, child exposure was affected by air and water contamination caused by pesticide drifts, runoff and leakage from the plantations, none of which were considered in the individual exposure indices. It is likely though that environmental exposures were similar for most children, since the plan-tations surrounded the entire village and most dwellings were close to the border of the plantations (Rojas et al., 2009). Likewise, we did not consider applications of agricultural and domestic use pesticides in homes in the ex-posure indices. It remains unclear whether all these factors combined di-rected the bias towards above or below true exposures. On the positive site, there was no interference from recent exposures since the outcome data were collected at a moment during which no agricultural activities were taking place. In our opinion kg of a.i is a better indicator of children’s exposure than hours of application, since farmers can relate directly the quantity of pesticide sprayed with the extension of cultivated area, whereas possibly some farmers report the total hours of the working day instead of hours of pesticide application. The association of cognitive and behavioral outcomes with pyrethroids exposure was reported with indices in hours of use not to
52
lose the sub-set of peanut plantation workers who reported high pyrethroid use and for whom the data about quantities applied was not available.
Recent exposures, as assessed through urinary metabolite levels, corre-spond to a particular moment in time. This means that determining the con-tribution of recent exposure to observed neurobehavioral effects is difficult since neurological impairment from pesticides tends to be chronic in nature. One indication that urinary pesticides residues may, up to a point, be a proxy for high usual exposures was the fact that the variability of TCPY and 3-PBA levels in this study were much higher between children than within children (in the 32 with repeated measurements).
Bias may also derive from the selection of the tests for outcome assess-ment, which followed the recommendations of an expert workshop (Anony-mous, 2006), where the expertise did not represent Nicaraguan rural condi-tions. It is known that the WICS-IV is highly influenced by social-cultural factors (Cronbach et al., 1990; Shuttleworth-Edwards et al., 2004), but since our outcome assessment included only participants from the same communi-ty with similar living conditions, the comparisons made within this study population are valid. Likewise, the Working Memory Index, where we found the strongest effects, has been documented as adequate for different cultures due to a low culturally acquired content need for performance on the digit span and letter-sequencing sub-tests (Wechsler, 2003; Shuttleworth-Edwards et al., 2004).
Strengths of this study worth noting are the high response rates (96% in 2007 and 100% in 2008) and the fact that all the participating families had similar socio-economic background and all children attended the same school, thereby reducing the possibility of confounding.
53
7. Conclusions
• The findings of this thesis demonstrated important long-term and recent pesticide exposures to highly toxic pesticides among chil-dren in an extremely poor population.
• Parental pesticide use influenced children’s exposure but envi-ronmental contamination was also an important contributor to to-tal children’s exposure.
• Prenatal as well as recent exposure to organophosphates influ-enced negatively children’s cognitive performance, in particular in relation to working memory, and total IQ. Exposure to pyrethroids in the first year of life influenced negatively children’s perfor-mance on perceptual reasoning, and increased hyperactivity and ADHD scales of behavior.
• Recent exposures to pyrethroids also seemed to influence chil-dren’s neurobehavioral functioning.
54
8. Implications
Given that almost half of Nicaraguan population lives in agricultural areas and the extensive use of pesticides for agricultural pest control, a large num-ber of children are potentially exposed to developmental neurotoxic sub-stances. Subsequently, the potential health effects in children are a major public health concern. This thesis adds to the growing body of evidence that exposure to pesticides early in the life, may result in cognitive and behavior-al deficits, these will negatively affect future achievements of the already socially disadvantaged children of this study.
Although agricultural workers received educational training through gov-ernmental institutions and some non-governmental organism, the health top-ics are usually around occupational acute poisoning prevention. It is neces-sary educational plans for reduce risky behaviors that increase the contact the whole family to the pesticides. On the other hand, increased efforts aimed at reducing pesticide are mandatory. The findings of this study should lead to regulatory action by the Nicaraguan government as well as interna-tional agencies to promote alternatives for pest control eliminating the risk of children pesticide exposure at source.
This thesis is the only one that associated neurobehavioral outcomes with long-term exposure in children. Although, a clear gap in the contribution of environmental contamination and other potential sources for children’s con-tact with pesticides exist, relevant associations were noted. Further research is necessary in order to refine the use of these indices for outcome assess-ment and more studies are necessary in order to explore potential effects in other organs and system highly vulnerable during the childhood such us endocrine and immune system.
55
9. Acknowledgements
I want to express my gratitude to everyone who in one way or another has contributed to this thesis: First of all, I want to thanks to children, parents, school teachers and community leaders of the study villages for their enthusiastic participation. Special thanks to Profesora Alba Luz Ramos and Sr. Lucas García for introduce me with the families. My main supervisor: Catharina (Ineke) Wesseling, for her scientific gui-dance, pushing me forward and for offered me a home environment during the writing process in Costa Rica and Finland. This thesis would not have been possible without her advices. My co-supervisors: Berna van Wendel de Joode for her guidance in exposure assessment and for valuable comments and Ingvar Lundberg for his comments, his support in paperwork and for his help during my stays in Sweden. Dr. René Altamirano and Dr. Armando Matute Deans of the Faculty of Me-dicine during my thesis period and my colleagues of the Physiological Sciences Department for provided me the time for dedicate to my thesis. To my collaborators in the Study 1: Matthew Keifer, Richard Fenske, Chensheng (Alex) Lu and Lisa Younglove, for their scientific collaboration, and their assistance during my stays in Seattle, Washington. Thanks to Ka-thlene Mirgon for administrative assistance, to Dr. José René Gasteazoro for introduce me with the banana plantation’s workers and to Aura Funez for assistance in samples handling and field work. To my collaborators in Study 2: Christian Lindh and Åsa Amilon for their support and assistance in laboratory analysis at Lund University; Marianela Rojas and Aurora Aragón for all the work done in the qualitative study, Dra. Ida Ana Alvir for training and supervision in neurological examination and David Hernández-Bonilla, for being my guru in the difficult art of the cognitive and behavioral assessment.
56
To my excellent field-work team: León García, Arlen Soto, Samaria Ba-lladares, José Ramón Avendaño, Lucía Solorzano and Guadalupe Loasiga. Thank you guys for work hard, for your commitment with the children and, just for being my friends. Also thanks to Marcos Vanegas, Axner Mayorga, Francisco Mayorga and José Angel Munguía our drivers during the field work. To my friend Cecilia Torres who is officially gone, but she is still alive in everything that she did. For all experiences shared in Nicaragua, Sweden and England when we started together this adventure of the PhD. To CISTA team, Edipcia Roque, Rogelio Caballero, Ana Yanci Ruiz, Ervin Esquivel, Leticia Mendez y Lilliam Medrano for their invaluable assistance in urine samples handling. Gladys Juárez, Marina Noguera, Martha Aragón, Mercedes Espinoza, Maritza Sevilla, Antonio Maravilla, Anan Pérez and Martha Moreno for administrative assistance and to my friend and co-lleagues Lylliam López, Freddy González, Luis Blanco, Indiana López, Marianela Corriols and Edmundo Torres for their advices and for their con-tribution to the occupational and environmental health in Nicaragua. To my friends in IRET, Costa Rica, Clemens Ruepert, Douglas Barraza, Rebeca Alvarado, Jennifer Crowe, Leonel Córdoba, Camilo Cano, Gicela Maldonado, Raquel Campos, Rocio Loria, Viria Bravo, Patricia Monge, Martha Castillo, Melvin Trejos, Rosario Quesada, Melania Chaverry and Diana Viquez for all memorable moments. To Timo Partanen, just for being who he is. Special thanks to Esperanza Gutiérrez (Doña Esperanza), Jamileth Miranda, and Ivette Freja Aleman for give me a little of Nicaraguan warm in the cold Sweden. Finally, I have to thanks this thesis to my family: To César Augusto, my husband, for his strong belief in me, for doing as single dad when I was away, for his love, encouragement and for just being there. My beloved daughters María Teresa and Ana Lucía for accepted to me as a part-time mother during my doctoral studies and for being my most important motivation. My parents Teresa and Antonio, my brother Antonio José, my sisters Mercedes, Ivania y Gioconda, my nephews Augusto, Fer-nando y Manuel and my niece María Fernanda for all your love and support. Of course this thesis would not have been possible without funding:
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Study 1 (Paper I) was supported by Fogarty International Center grant 5 D43 TW00642, and by the U.S. EPA through STAR grant R-828606 to the Uni-versity of Washington. Additional support was provided through the Re-search Department of the Swedish International Development Agency (Sida/SAREC), and the NIOSH Agricultural Centers Program (5 U50 OH07544). Study 2 (Papers II to IV) was supported by Sida/SAREC (Swedish Interna-tional Development Agency) and Formas (Swedish Research Council).
I come from the so-called third world. Which is the second?
Vengo del llamado tercer mundo. ¿Cuál es el segundo?
Isabel Allende
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“La única manera de detener el uso de venenos, es si otras fuentes de empleos existan
porque sin estas, si no nos morimos por el veneno, nos vamos a morir de hambre”
Una madre de Los Zanjones, Posoltega, Nicaragua
Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 820
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine.
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2012
